investigation of magnesium ions effect on sludge...
TRANSCRIPT
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INVESTIGATION OF MAGNESIUM IONS EFFECT ON SLUDGE PROPERTIES IN PHOSPHORUS DEFICIENT
BIOREACTORS
A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES
OF MIDDLE EAST TECHNICAL UNIVERSITY
BY
EDA ÜNAL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE IN
BIOTECHNOLOGY
SEPTEMBER 2010
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Approval of the thesis:
INVESTIGATION OF MAGNESIUM IONS EFFECT ON SLUDGE
PROPERTIES IN PHOSPHORUS DEFICIENT BIOREACTORS
submitted by EDA ÜNAL in partial fulfillment of the requirements for the degree of Master of Sciences in Biotechnology Department, Middle East Technical University by,
Prof. Dr. Canan Özgen . . Dean, Graduate School of Natural and Applied Sciences Prof. Dr. İnci Eroğlu . Head of the Department, Biotechnology Prof. Dr. F. Dilek Sanin . Supervisor, Environmental Engineering Dept., METU . Prof. Dr. G. Candan Gürakan . Co-supervisor, Food Engineering Dept., METU
Examining Committee Members: Prof. Dr. Pınar Çalık . Chemical Engineering Dept., METU Prof. Dr. F. Dilek Sanin . Environmental Engineering Dept., METU . Prof. Dr. G. Candan Gürakan . Food Engineering Dept., METU Assoc. Prof. Ayşegül Aksoy . Environmantal Engineering Dept., METU Dr. Tuba Hande Ergüder . Environmental Engineering Dept., METU Date:.
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I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original in this work.
Name, Last name: Eda ÜNAL
Signature
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ABSTRACT
INVESTIGATION OF MAGNESIUM IONS EFFECT ON SLUDGE
PROPERTIES IN PHOSPHORUS DEFICIENT BIOREACTORS
Ünal, Eda
M.S., Department of Biotechnology
Supervisor: Prof. Dr. F. Dilek Sanin
Co-Supervisor: Prof. Dr.G. Candan Gürakan
September 2010, 122 pages
The activated sludge process efficiency depends on separation of microbial cells
from treated wastewater. Separation can fail due to a number of problems. One of
these problems is sludge bulking which is non-settling situation of biomass. Former
studies showed that phosphorus deficiency caused filamentous sludge bulking with
increasing magnesium ion concentrations. The main objectives of this study are to
find out the effect of magnesium ions on sludge properties in phosphorus deficient
medium and to determine if there is any bulking. Three different concentrations of
magnesium (0.5, 5, 15 meq/L) were added to three bioreactors which contained
phosphorus deficient medium. In first set C: N: P ratio was 100:5:0.05. In second set,
C:N:P ratio was elevated to 100:5:1. At steady state, physical characteristics
including sludge volume index (SVI), viscosity, turbidity and dewaterability were
determined. Besides concentration of extracellular polymeric substances (EPS) as
well as conductivity was measured. By using API kits, bacterial identification was
achieved.
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In first set phosphorus deficiency and increasing magnesium ion concentration
caused filamentous bulking. Carbohydrate content of extracellular polymeric
substance significantly increased by magnesium addition. Dewaterability of the
system got worse and viscosity decreased. Sludge Volume Index (SVI) indicated
severe bulking at all magnesium concentrations. By using biochemical tests
microorganisms dominant in the system were determined
In second set, all of the parameters indicated healthy flocculation. By magnesium
addition, EPSp and EPSc increased. Dewaterability and settleability, improved by the
presence of phosphorus with close values measured at different magnesiuım
concentrations. Nocardia related genera of Corynebacterium and Enteric
microorganisms were identified.
Keywords: Activated sludge, magnesium, filamentous sludge bulking, extracellular
polymeric substances, phosphorus
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ÖZ
MAGNESYUM İYONLARININ FOSFOR YETERSİZLİĞİ OLAN
BİYOREAKTÖRLERDE ÇAMUR ÖZELLİKLERİNE OLAN ETKİSİNİN
İNCELENMESİ
Ünal, Eda
Yüksek Lisans, Biyoteknoloji Bölümü
Tez Yöneticis: Prof. Dr. F. Dilek Sanin
Ortak Tez Yöneticisi: Prof Dr. G. Candan Gürakan
Eylül 2010, 122 sayfa
Aktif çamur işleminin verimliliği mikrobiyal hücrelerin arıtılmış atıksudan
ayrılmasına bağlıdır. Ayrılma birtakım problemler nedeniyle başarısız olabilir. Bu
problemlerden birisi biyokütlenin çökmemesi durumu olan çamur şişmesidir. Daha
önceki çalışmalar fosfor yetersizliğinin, artan magnesyum iyonu
konsantrasyonlarıyla birlikte filamentli çamur şişmesine neden olduğunu
göstermiştir. Bu çalışmanın ana hedefleri fosfor eksikliği olan besi ortamında
magnesyum iyonlarının çamur özelliklerine olan etkisini ortaya çıkarmak ve çamur
şişmesi olup olmadığını belirlemektir. Fosfor eksik besi ortamı içeren üç
biyoreaktöre üç farklı konsantrasyonda magnesyum (0.5, 5, 15 meq/L) ilave
edilmiştir. Ilk aşamada KOİ/N/P oranı 100:5:0.05 olmuştur. İkinci aşamada,
KOİ/N/P oranı 100:5:1’ ye yükseltilmiştir. Kararlı halde, çamur hacim indeksini
(SVI), viskositeyi, bulanıklığı ve susuzlaştırılabilirliği içeren fiziksel
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özellikler belirlenmiştir. Bunlarla beraber iletkenlikte olduğu gibi hücre dışı
polimerik madde (HDP) konsantrasyonu da ölçülmüştür. API kitleri
kullanarak bakteriyel tanımlanma başarılmıştır.
İlk sette, fosfor yetersizliği ve artan magnesyum iyonu konsantrasyonu, filamentli
çamur şişmesine neden olmuştur. Hücredışı polimerik maddelerin (HDP)
karbonhidrat içeriği, magnezyum eklenmesiyle önemli ölçüde artmıştır. Sistemin
susuzlaştırılabilirliği kötüleşmiş, viskozite düşmüştür. Çamur hacim indeksi (ÇHİ)
bütün magnezyum konsantrasyonlarında ciddi çamur şişmesini göstermiştir.
Biyokimyasal testleri kullanarak, sistemdeki baskın mikroorganizmalar
belirlenmiştir.
İkinci aşamada bütün parametreler , sistemin sağlıklı yumaklaşmasını gösterir
şekilde iyileşmiştir. Magnezyum iyonu eklenmesiyle HDP’nin hem protein hem de
karbohidrat içeriği artmıştır. Sistemin çökebilirliği ve susuzlaştırılabilirliği, değişik
magnezyum konsantrasyonlarında yakın değerler elde edilecek şekilde iyileşmiştir.
Enterik mikroorganizmalar ve Nocardia ile alakalı Corynebacterium cinsi
mikrorganizmalar belirlenmiştir.
Anahtar kelimeler: Aktif çamur, magnezyum, filamentli çamur şişmesi, hücre dışı
polimerler, fosfor
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ACKNOWLEDGEMENTS
I would like to express my high appreciation and faithful gratitude to my supervisor,
Prof. Dr. F. Dilek Sanin for her valuable guidance, suggestion, patience and
encouragement through the time of the study and in the preparation of this thesis.
Also, I would like to thank my co-supervisor, Assoc. Prof. Dr. G. Candan Gürakan,
for the encouragement, knowledge and support throughout the research period.
I would also like to thank the dissertation committee members Prof. Dr. Pınar Çalık,
Assoc. Prof. Dr. Ayşegül Aksoy and Assoc. Prof. Dr. Tuba Hande Ergüder for their
guidance, suggestions and contributions.
I would like to thank to the all members of Department of Environmental
Engineering of Middle East Technical University. I would also greatful to Mehmet
Dumanoğulları and Gizem Uğurlu Turan for their technical assistance.
I am thankful to Fadime Kara and Çiğdem Kıvılcımdan Moral who have trained me
in laboratory experiments conducted in this study for their kindness, patient and
support. They are the ones that always encouraged me during all the processes of my
thesis study. I also would like to thank to my friends, Ceren Aksu, Saniye Keser,
Nurhan Şen, Kerem Talu, Abdullah Öğütverici, İrem İpçi and Müneer Ahmed for
their endless help, encouragement and support during this research. I shared
everything including laughs, exhaustion, happiness, hope, despair etc. Thank you for
always being near me whenever I needed. Lastly I would like to dedicate the thesis to
my parents Erçin and Ayşin Ünal and my sister Seda Ünal who are always
supporting me for everything that I have done in my life. I would like to express my
deepest appreciation to them for their endless love, encouragement, confidence, and
patience not only during this study but also throughout my life.
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TABLE OF CONTENTS
ABSTRACT ........................................................................................................... iv
ÖZ .......................................................................................................................... vi
ACKNOWLEDGEMENTS ................................................................................. viii
TABLE OF CONTENTS ........................................................................................ ix
LIST OF TABLES ............................................................................................... xiii
LIST OF FİGURES ............................................................................................. xiv
CHAPTER
1 INTRODUCTION ................................................................................................ 1
2.LITERATURE REVIEW ...................................................................................... 4
2.1 Activated Sludge System and Its Components ........................................ 4
2.2 Bioflocculation And Its Mechanisms ...................................................... 7
2.2.1. Bioflocculation ................................................................................. 7
2.2.2.BioflocculationMechanisms ............................................................... 8
2.2.2.1. The Zoogloea Ramigera Theory ................................................ 8
2.2.2.2. Flagella Agglutination Theory and Protozoa Theory .................. 8
2.2.2.3. PHB (poly-beta-hyroxybutryric acid) Theory ............................ 9
2.2.2.4. Extracellular Polymeric Substances and Bioflocculation ........... 9
2.2.2.5. Filament Backbone Theory........................................................ 9
2.2.2.6. Double Layer Theory (DLVO Theory) ....................................10
2.2.2.7. Polymer Bridging Model .........................................................11
2.2.2.8. Divalent Cation Bridging Theory (DCB) .................................12
2.2.2.9. Alginate Theory .......................................................................12
2.3 EPS .......................................................................................................14
2.3.1 Definition of EPS .............................................................................14
2.3.2 Composition of EPS .........................................................................15
2.3.3 Factors Affecting EPS Production and Bioflocculation….………..…….16
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2.4 Cations ..................................................................................................18
2.4.1 Divalent Cations ...............................................................................19
2.4.2 Monovalent Cations ..........................................................................21
2.4.3 M/D Ratio ........................................................................................22
2.4.4 Trivalent Cations ..............................................................................22
2.5 Activated Sludge Properties Related To Bioflocculation ........................23
2.5.1 Dewaterability ..................................................................................23
2.5.2 Settleability ......................................................................................27
2.5.3 Rheology ..........................................................................................29
2.6 Bulking ................................................................................................ .32
2.6.1 Filamentous Bulking .................................................................... ….33
2.6.2 Zoogleal Bulking .............................................................................35
3.MATERIALS AND METHODS..........................................................................37
3.1 Experimental Set-Up And Reactor Operation ........................................37
3.1.1. Phosphorus Deficient Conditions ....................................................38
3.1.2. Phosphorus Sufficient Conditions ....................................................40
3.2 Analysis Conducted At Steady State .....................................................41
3.2.1. Chemical Analyses Conducted At Steady State ................................41
3.2.1.1. EPS Extraction and Protein Carbohydrate Analysis ..................41
3.2.1.1.1. EPS Extraction ....................................................................41
3.2.1.1.2. Carbohydrate Analysis ........................................................43
3.2.1.1.3. Protein Analysis ..................................................................44
3.2.1.2. Conductivity ............................................................................44
3.2.2. Physical Analyses Conducted At Steady State .................................45
3.2.2.1 Viscosity ...................................................................................45
3.2.2.2. Sludge Volume Index (SVI) ....................................................45
3.2.2.3. Turbidity ..................................................................................46
3.2.2.4. Capillary Suction Time (CST) .................................................46
3.2.3. Microbiological Analyses Conducted At Steady State .....................47
3.2.3.1. Analysis Conducted for Bacterial Identification........................48
3.2.3.2. Quantification of Bacteria of the Wastewater Sludge ..............48
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3.2.3.3. Additional Tests Required for Using The Appropriate API Test
.............................................................................................................50
3.2.3.3.1. Catalase Test ......................................................................50
3.2.3.3.2. Gram Staning .....................................................................50
3.2.3.3.3. Oxidase Test ........................................................................51
3.2.3.4. The API 20E System ...............................................................51
3.2.3.5. API Coryne System ..................................................................52
3.2.4. Other Measurements ........................................................................53
3.2.4.1. MLSS and MLVSS ..................................................................53
3.2.4.2. COD ........................................................................................54
3.2.4.3. pH ............................................................................................54
4.RESULTS AND DISCUSSION ...........................................................................55
4.1 Results Of The Reactors At Phosphorus Deficient Conditions ...............55
4.1.1 Results of the Chemical Analyses .....................................................56
4.1.1.1 Results of COD Measurements .................................................56
4.1.1.2 Results of the Extracellular Polymeric Substance Extraction,
Carbohydrate and Protein Analysis .......................................................57
4.1.1.3 Conductivity Results .................................................................59
4.1.2 Results of the Physical Analyses .......................................................60
4.1.2.1 Settleability Results ...................................................................60
4.1.2.2 Dewaterability Results ..............................................................62
4.1.2.3 Rheology ...................................................................................64
4.1.2.4 Turbidity Results .......................................................................68
4.1.3 Results of the Microbiological Analyses ...........................................69
4.2 Results Of The Reactors At Phosphorus Sufficient Conditions ..............74
4.2.1 Results of the Chemical Analyses ....................................................75
4.2.1.1 Results of COD Measurements ..................................................75
4.2.1.2 Results of the Extracellular Polymeric Substance Extraction,
Carbohydrate and Protein Analysis .......................................................75
4.2.1.3 Conductivity Results .................................................................77
4.2.2 Results of the Physical Analyses ......................................................78
4.2.2.1 Settleability Results ...................................................................78
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4.2.2.2 Dewaterability Results ..............................................................79
4.2.2.3 Rheology Results ......................................................................80
4.2.2.4 Turbidity Results .......................................................................84
4.2.3 Results of the Microbiological Analyses ...........................................85
4.3 .. Comparison of the Phosphorus Deficient and Phosphorus Sufficient Conditions
...............................................................................................................................88
4.3.1 COD Results ..........................................................................................88
4.3.2 Results of the Extracellular Polymeric Substance Extraction, Carbohydrate
and Protein Analyses...............................................................................................89
4.3.3. Conductivity Results .............................................................................90
4.3.4 Settleability Results ...............................................................................91
4.3.5 Dewaterability Results ..........................................................................92
4.3.6 Viscosity Results ...................................................................................93
4.3.7 Microbiological Analyses ......................................................................94
5. CONCLUSION ...................................................................................................96
6.RECOMMENDATIONS .....................................................................................99
REFERENCES ..................................................................................................... 100
APPENDICES
APPENDIX A Solids Concentrations of Reactors under Deficient Phosphorus and
Sufficient Phosphorus Conditions………..……………...…………………..….109
APPENDIX B Calibration Curves for EPS Analyses...............................….......115
APPENDIX C Api Test Results…………………….…......................................117
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LİST OF TABLES
Table 3.1 the Composition of the Synthetic Feed Medium Given to the Reactors 39
Table 3.2 the Composition of the Synthetic Feed Medium Given to the Reactors 40
Table 4.1 CST Values with respect to Magnesium Ion Concentration…............ 62
Table 4.2 total heterotrophic bacteria count with respect to magnesium ion
concentration.......................................................................................................... 72
Table 4.3 Results of API 20E biochemical kit…………………….........................74
Table 4.4 CST Values with respect to Magnesium Ion Concentration…………... 80
Table 4.5 Total heterotrophic bacteria count results…………………………..… 87
Table 4.6 Results of API 20E…………………………………………………… 87
Table 4.7 Results of API Coryne………………………………………………… 88
Table 4.8 Effluent COD concentration in phosphorus deficient and phosphorus
present conditions……………………………………………………………… 89
Table 4.9 Composition and concentration of EPS under phosphorus deficient and
phosphorus present conditions…………………………………………………… 90
Table 4.10 Conductivity values under sufficient and deficient phosphorus
concentration……………..……………………………………………………….91
Table 4.11 SVI values with respect to magnesium concentration under phosphorus
deficient and phosphorus present conditions……………………………………... 92
Table 4.12 Normalized CST values with respect to magnesium concentration under
phosphorus deficient and phosphorus present conditions……………………….. 93
Table 4.13 Apparent viscosities at different magnesium concentrations under
phosphorus deficient and phosphorus present conditions……………………….. 94
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LIST OF FİGURES
Figure 2.1 Schematic Representation of Waste Treatment System with Activated
Sludge Components………………………………………………………..………4
Figure 2.2 Depiction of the Filament Backbone (Sezgin et al., 1978)………… 10
Figure 2.3 Demonstration of the Double Layer……………………………….. 11
Figure 2.4. Depiction of the DCB Theory…………………………….………. 12
Figure 2.5 Roles of Biopolymers and Divalent Cations on Bioflocculation
(B:Bacteria, LLP:Lectin-like protein,P:polysaccharide,C++:Divalent cation (jenkins
et al,2006)…………………………………………………………………….. 19
Figure 2.6 The Rheograms of Different Fluids(Vesilind,1979)………………. 30
Figure 3.1. Schematic Representation of the Reactor Set-Up……………..….. 38
Figure 3.2 a) The Basic Appearance of a CST Device (Vesilind, 1988) b) Part of the
Triton Electronics Type 304M CST with its Stainless Steel Collar, Plastic Blocks
with Contact Points of Them…………………………………………………… 47
Figure 3.3 Streaking Process…………………………………………………… 49
Figure 3.4 API 20E Kit Apparatus…………………………………………….. 51
Figure 3.5 API Coryne Apparatus…………………………………………..….. 53
Figure 4.1 Effluent COD Values versus Magnesium Ion Concentration…..….. 57
Figure 4.2 EPSP, EPSc and total EPS Concentration Versus Magnesium Ion
Concentration……………………………………………………………….…. 58
Figure 4.3 Conductivity of the Sludge with respect to Magnesium Ion Concentration
Provided in the Feed………………………………………………………….… 60
Figure 4.4 SVI Values at Different Magnesium Concentrations……………… 61
Figure 4.5 Normalized CST Measurements with Respect to Magnesium Ion
Concentration………………………………………………………………… 64
Figure 4.6 Typical Rheograms for a. Control Reactor at 1851 mg/L b.5meq/L
Reactor at 2260 mg/L c.15meq/L Reactor at 2800 mg/L MLSS Concentration... 66
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Figure 4.7 Apparent Viscosity Values with Respect to Magnesium Ion
Concentration……………………………………………………………………. 67
Figure 4.8 Apparent Viscosities versus Magnesium Ion Concentration at a Fixed
MLSS Concentration of 1500mg/L…………………………………………….. 67
Figure 4.9 Effluent Turbidity Values versus Magnesium Ion Concentration…. 69
Figure 4.10 Photomicrographs of control reactor containing 0.5meq/L Mg under
4 × magnification…………………………………………………………….. 70
Figure 4.11 Photomicrographs of reactor containing 5meq/L Mg, under 40�
Magnification………………………………………………………………….. 70
Figure 4.12 Photomicrographs of reactor containing 15meq/L Mg, under 4 �
Magnification……………………………………………………………….. 71
Figure 4.13 Effluent COD Values versus Magnesium Ion Concentration…… 75
Figure 4.14 EPSP, EPSC and total EPS Concentration Versus Magnesium Ion
Concentration…………………………………………………………………. 76
Figure 4.15 Conductivity of the Sludge with Respect to Magnesium Ion
Concentration Provided In the Feed…………………………………………… 78
Figure 4.16. SVI Values at Different Magnesium Concentrations……………. 79
Figure 4.17 Normalized CST Measurements with Respect to Magnesium Ion
Concentration…………………………………………………………………. 80
Figure 4.18 Typical Rheograms for a. Control reactor at 2331mg/L b.5meq/L reactor
at 2296mg/L c.15meq/L reactor at 1946mg/L MLSS Concentration……….. 82
Figure 4.19 Apparent Viscosity Values with Respect to Magnesium Ion
Concentration………………………………………………………………… 83
Figure 4.20 Apparent Viscosity vs Magnesium Ion Concentration in the Fixed MLSS
Concentration of 1500 mg/L…………………………………………………. 83
Figure 4.21 Effluent Turbidity Values versus Magnesium Ion Concentration… 84
Figure 4.22 Photomicrographs of control reactor containing 0.5meq/l Magnesium
under 4 × magnification………………………………………………………. 85
Figure 4.23 Photomicrographs of Reactor Containing 5meq/l Magnesium under 4 ×
Magnification…………………………………………………………………… 85
Figure 4.24 Photomicrographs of reactor containing 15meq/l Magnesium under under 4 × Magnification……………………………………………………… 86
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LİST OF ABBREVIATIONS
BSA : Bovine Serume Albumin
CER : Cation exchange resin
CGY : Casitone-glycerol-yeast extract
C/N : Carbon to nitrogen ratio
C/N/P : Carbon to nitrogen to phosphorus ratio
COD : Chemical oxygen demand
CST : Capillary suction time
DO : Dissolved oxygen
EDTA : Ethylenediaminetetraacetic acid
EGTA : Ethylene glycol tetraacetic acid
EPS : Extracellular polymeric substances
EPSc : Carbohydrate constituent of EPS
EPSp : Protein constituent of EPS
MLSS : Mixed liquor suspended solids
MLVSS : Mixed liquor volatile suspended solids
NTU : Nephelometric turbidity unit
PBS : Phosphate buffer saline
SRF : Specific resistance to filtration
SVI : Sludge volume index
TKN : Total kjeldahl nitrogen
VSS : Volatile suspended solids
ZSV Zone settling velocity
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CHAPTER 1
INTRODUCTION
Activated sludge systems are known as the most conventional and popular method
among several biological treatment processes. It is composed of aeration and further
settling of microbial mass. Organic substrate is utilized and converted to carbon
dioxide, water and new biomass in the aeration tank. After this utilization step,
proper settling of the new biomass is essential for the efficiency of the treatment.
Separation of microorganisms from the effluent is crucial. This separation and
settling are highly dependent on the healthy bioflocculation of microbial species.
Bioflocculation plays a vital role in this process (Pavoni et. al, 1972). As a result of
this significance there are number of researches that focused on the dominant
mechanism of bioflocculation or the microbial aggregation (Higgins et al., 2002).
Several theories for the bioflocculation were proposed over the years.
Floc structure in activated sludge systems is mainly composed of microorganisms,
cell debris, cations and extracellular polymeric substances (EPS) (Eriksson and Alm,
1991; Bruus et al., 1992; Higgins and Novak, 1997 a, b). Among them extracellular
polymeric substances and cations were proposed to have interactions in order to
contribute to the bioflocculation mechanisms. Due to the negative charge carried by
the majority of EPS, cations are crucial for the floc structure in order to provide
bridging to the negative sites on biopolmer network (Bruus et al., 1992; Urbain et al.,
1993; Higgins and Novak, 1997a).
There are several researches in literature that dealt with the effect of cations on
bioflocculation mechanism. However some of them could not reveal ultimate effect
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of cations on flocculation due to the operation in batch mode (Higgins and Novak,
1997, a, b). In addition some studies investigated the effect of cations on the system
having only mono culture bacteria. Since activated sludge system is composed of
mixed cultures that contribute discrepant properties to floc structure, the results with
monocultures could not reflect actual case (Kara, 2007).
Many researchers have focused on the effect of divalent cations in bioflocculation
and therefore physical and chemical characteristics of the system. Higgins and
Novak in 1997, proposed a floc model with greater contribution of the divalent
cations such that lectin like proteins that were attached to bacterial surface were
cross-linked with polysaccharides by the help of calcium and magnesium. The study
conducted by Bruus et al. in 1992, indicated that the extracellular polymers may be
alginate or another polysaccharide properties of which resemble to alginate and form
a gel-like structure in the presence of calcium ions. Sanin and Vesilind in 1996,
supported this proposal by the establishment of synthetic sludge flocs due to the
addition of calcium and alginate. Higgins and Sobeck in 2002 found that sludge
settling and dewaterability characteristics enhanced by addition of either magnesium
or calcium.
Binding ability of the components of the extracellular biopolymers to divalent
cations was studied as another issue in some other research. Calcium and magnesium
were found to increase bound protein concentration of EPS (Urbain et al., 1993;
Dignac et al., 1998, Higgins and Novak, 1997, a).
It is known from the literature that flocculation of the microorganisms is dependent
on many factors. Among them one of the most important is the concentration of the
nutrients that is provided in the feed. Under nutrient deficient conditions
macrostructure failure of the system which is called as sludge bulking occurs
(Jenkins et al., 1993). In addition to this, sludge bulking can differ in type with
addition of different cations under nutrient deficient conditions. For instance under
phosphorus deficiency with abundance of magnesium in the feed, filamentous
bulking was reported (Turtin, 2005) whereas viscous bulking was observed under
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phosphorus deficiency and calcium ion abundance in the system (Vatansever, 2005).
However these studies were not detailed enough to identify all components related to
bioflocculation.
This study is conducted to investigate the effect of magnesium addition on
bioflocculation and sludge bulking at phosphorus deficient and phosphorus sufficient
conditions. The semi-continuous reactors were used in order to reflect the effect of
divalent cation addition more accurately than batch reactors. Physical, chemical and
microbiological analyses were conducted during the study. It is desired to know
under sludge bulking conditions if the improvement of flocculation can be achieved
or not with divalent cation addition to the system. Besides, under phosphorus
sufficient conditions the effect of magnesium was studied in the second set. By this
way comparison of two systems could be done.
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CHAPTER 2
LITERATURE REVIEW
2.1 Activated Sludge System and Its Components
One of the most common and conventional methods of the wastewater treatment is
known as activated sludge process. This process contains several metabolic reactions
such as synthesis, nitrification and respiration of the microorganisms; separation and
settling of activated sludge solids; removing excessive amount of the sludge to be
further processed in thickeners and recycling the required amount of the
microorganisms back to the system.
The schematic representation of such a system can be seen in Figure 2.1 below. After
the pre-treatment reactor, activated sludge process begins at aeration zone and
continues at the settling tank.
Figure 2.1 Schematic Representation of Waste Treatment System with Activated
Sludge Component
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In the aeration tank, the air is introduced to the system in order to achieve aerobic
conditions and mixing. This step involves the utilization of colloidal and suspended
organic material by the microorganisms. The products are mainly carbon dioxide,
water and new biomass. In addition to this, the microorganisms convert ammonia
nitrogen to nitrate nitrogen by the nitrification process. The efficiency of first step or
substrate utilization phase depends on providing required environmental conditions
to active biomass.
After the aeration tank, wastewater comes to secondary clarifier. In this step, main
aim is to obtain clear supernatant by providing the effective settling of produced
biomass which completed its metabolic role in the first step, other suspended and
colloidal components. The effectiveness depends on the successful biosolids/liquid
separation by bioflocculation and solids/liquid separation. Therefore bioflocculation
plays a vital role in this process (Pavoni et. al, 1972).
After successive separation of the biomass from the supernatant, some of the settled
sludge/biomass is returned to the beginning of the aeration tank in order to sustain
same F/M. Moreover, some of the settled and excess biomass is wasted from the
system in order to be handled in the thickeners.
Although all of the process can be seen as physical and chemical process,
microorganisms play a major role in substrate utilization and settling steps of the
activated sludge. Among the heterogeneous mixture of activated sludge that is
composed of particles, colloids, organic polymers and cations, there is also
microorganisms (Jorand et al., 1995). As the biological components, system is
comprised of bacteria, fungi, protozoa and rotifers. The dominance of the
microorganism depends primarily on the environmental conditions, wastewater
characteristics, process design and mode of plant operation (UCLA College of
Letters and Science, 2010).
Although there are number of the microorganisms present, most recognizable one in
the system is bacteria. The majority of the bacteria in the system are facultative, able
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6
to live in the presence or absence of oxygen. In addition, the system is highly
heterotrophic. Arthrobacter, Citromonas, Flavobacterium, Pseudomonas,
Alcaligenes, Achromobacter is some of the genera that can be found in activated
sludge (Jenkins et al., 1993). Besides this, autotrophic bacteria which utilize
ammonia nitrogen to nitrate nitrogen, found in smaller amounts compared to
heterotrophic bacteria. This may be due to the slower growth rate of these types of
bacteria. Most common autotrophic bacteria are identified as Nitrobacteria and
Nitrosamonas (UCLA College of Letters and Science, 2010).
Besides several species of the bacteria there are also eukaryotic organisms in
activated sludge process. One of these organisms is fungi most of which are strictly
aerobe and can resist to low pH and nitrogen deficiency. Excessive proliferation of
filamentous fungi was reported to cause settleability problems in settling tanks of
activated sludge systems. Some species of the protozoa also have been identified in
activated sludge. They are the indicators of oxygen availability in the system due to
their aerobic features. However some types such as amoebae and flagellates are able
to resist to anerobic conditions (Gerardi, 2006). Rotifers which are multicellular
organisms are strictly sensitive to oxygen depletion and toxicity as well as the
protozoa. They are found in a stable activated sludge environment and they seem as a
recirculating wheel when they move (UCLA College of Letters and Science, 2010).
Aggregation of all these organisms is essential in the second step of the system.
Activated sludge structure is composed of three micro-structures: bacteria, micro-
colonies and flocs (Jorand, et al., 1995; Snidaro et. al, 1997). First of all
microorganisms or specifically bacteria stick to each other by a polymeric matrix and
form the second level, micro-colonies. Then extracellular polymeric substances and
cations bridge separate micro-colonies and form the final step, activated sludge flocs.
The flocculated microbial aggregates called as flocs change in size from 10 to 1000
μm (Andreadakis, 1993).
According to Jenkins et al., (1993), floc structure is composed of macrostructure and
microstructure. Microstructure is formed by microbial aggregation, bioflocculation
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7
and adhesion. Macrostructure is composed of filamentous microorganisms that form
the backbone of the floc. Main floc forming heterotrophic bacteria are listed as
Achromobacter, Alcaligenes, Arthrobacter, Citromonas, Flavobacterium,
Pseudomonas, and Zoogloea.
2.2 Bioflocculation and Its Mechanisms
2.2.1. Bioflocculation
Bioflocculation which can be defined as the microbial aggregation is crucial for the
effluent quality in biological wastewater treatment systems. The microbial
aggregation takes place by the floc formation of microorganisms with the help of
microbially produced extracellular polymeric substances. When the microbial
aggregation is not achieved successfully, the solid/liquid separation in the secondary
clarifier becomes ineffective and leads to the problems in dewaterability and settling.
Due to decrease in dewaterability of the system the further removal of the water by
thickening and other mechanical means become impossible. In addition, the main
purpose of the treatment process becomes useless due to the settling problems. Due
to this high significance in the overall process several researches have been
performed for understanding the driving mechanisms of bioflocculation (Sobeck &
Higgins, 2002).
Progresses that can be achieved in understanding the whole picture behind the
microbial aggregation mechanisms have great importance in the biological
wastewater treatment which is not limited to activated sludge process. These systems
include:
• aerobic waste treatment containing the bacteria and other microorganisms;
• anaerobic waste treatment containing the bacteria and other microorganisms;
• stabilization ponds and nutrient-stripping processes containing algae and
other microorganisms and
• the dewatering process of excess biomass (such as bacteria and algae)
synthesized in the above types of biological treatment (Pavoni et al, 1972).
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8
2.2.2. Bioflocculation Mechanisms
There have been several studies conducted in order to understand the major
mechanism in the bioflocculation process for years. According to these studies, some
of the bioflocculation theories have been proposed.
2.2.2.1. The Zoogloea Ramigera Theory
The first theory in this field is known as Zooglea Ramigera Theory which was
studied by C.T Butterfield in 1935, H. Heukelekian and coworkers in 1939. They
claim that bioflocculation ability of the activated sludge systems is dependent on the
particular group of bacteria Zoogloea Ramigera due to their extracellular gelatinous
matrix which keeps the microbial aggregates together. However the validity of the
theory was lost by the further studies indicating that other bacterial species which
were isolated from the activated sludge environment was able to make flocs (Mc
Kinney, 1952; McKinney & Horwood, 1952; McKinney & Weichlein, 1953).In
addition, majority of the scientists is in agreement that Zoogleal growths do not
involve in microbial aggregation all the time (Friedman & Dugan, 1968).
2.2.2.2. Flagella Agglutination Theory and Protozoa Theory
In 1938, A. Pijper proposed that flagella interactions may be the reason for the
agglutination of bacteria. However for the species not having flagellar interactions,
bioflocculation occurred generally. In the same manner, protozoa were thought as a
supporter for the microbial aggregation (Pillai, 1941; Barker, 1946). As in the case of
flagella it was proved that many pure cultures were able to form flocs. Therefore,
these two proposals seem to be limited and not inclusive rather than being
completely wrong.
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9
2.2.2.3. PHB (poly-beta-hyroxybutryric acid) Theory
K.Crabtree and his co-workers in 1965 and in 1969 considered PHB in the cells as
the direct constituent responsible for the microbial aggregation. A study proposed the
presence of PHB granules in some of the cells during bioflocculation although the
direct correlation between aggregation and PHB could not be found (Friedman et al.,
1968). Many researchers mainly focused on the food reserving function of PHB
which can be used during endogenous growth rather than bioflocculation potential
(Macrae & Wilkinson, 1958; Rouf & Stokes, 1962).Therefore there was not a direct
correlation between flocculation and PHB content of the cell and can not be
considered as major bioflocculation mechanism.
2.2.2.4. Extracellular Polymeric Substances (EPS) and Bioflocculation
Several bioflocculation mechanisms are based on the formation of EPS. From the
past research there appears to be a close relationship between bioflocculation and
EPS. This relationship was firstly studied by R. E. Mc Kinney in 1953. He stated that
the cell wall enveloped by polysaccharide could play a role in reduction of the
surface potential and could increase the aggregation.
2.2.2.5. Filament Backbone Theory
The theory which was first proposed by D. S. Parker and his co-workers in 1972
necessitates the presence of filaments in bioflocculation. Once the microorganisms
attach to each other and form flocs by the presence of polymer bridges, these
attachments then bind to filaments. The floc strength becomes highly dependent on
these filaments in a way that filaments constitute the backbone of the aggregation.
The schematic presentation of filament backbone is seen in Figure 2.2 below
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10
Figure 2.2 Depiction of the Filament Backbone (Sezgin et al., 1978)
2.2.2.6. Double Layer Theory (DLVO Theory)
Double Layer Theory, developed by Derjaguin, Landau, Verwey and Overbeek, can
be regarded as the classical colloidal theory that assumes presence of electrical
double layer around charged particles. In an electrolytic solution, oppositely charged
ions are attracted to the surface of the charged particle therefore get closer;
oppositely charged ions can either be distributed in different parts of the solution
(Gregory, 1992). Around the charged particles there has been the formation of
diffuse cloud of ions due to electrostatic attraction and ionic diffusion. This cloud of
ions is described as the electrical double layer (Kruyt, 1952). Double layer is mainly
composed of two layers, Stern layer and Diffuse layer. Double layer is demonstrated
in Figure 2.3. The counter-ions are strongly attached to the surface of the charged
particle and forms Stern layer. The outer layer, named as Diffuse layer, contains
counter-ions which are repelled by the Stern layer’s counter ions. The attraction by
the particle continues but repulsion is stronger and hence these counter ions seem
less tightly associated with the particle (Adamson, 1990). The density of the ions in
the diffused layer reduces going through the bulk liquid until it becomes equivalent
to that of bulk liquid. Because of the presence of double layer and hence repulsion,
the adjacent particles can not coalescence with the charged particle.
When the ionic strength increases, the double layer size decreases and hence the
attraction of other particles increases. From this theory it can be concluded that by
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11
the addition of the cations to activated sludge systems, the double layer compression
takes place and bioflocculation is favored. This idea was supported by some of the
researchers. C. P. Cousin and J. J. Gnaczarcyzk in 1998, studied sodium effect; found
out an increase in floc size and deduced it to working of this theory. In addition, A.
Zita and M. Hermansson in 1994 found the association of floc stability with ionic
strength and DLVO theory and studied the effect of calcium and potassium ions.
Figure 2.3 Demonstration of the Double Layer
2.2.2.7. Polymer Bridging Model
According to polymer bridging theory, long-chain polyelectrolyte with high
molecular weight, support the interaction of bacterial cells, other individual cells and
particles by acting just as a bridge and hence cause aggregation of bacteria. In order
to achieve flocculation net electrostatic surface charge should be equal to zero
(Tenney & Stumm, 1965; Ries & Meyers, 1968). However one of the studies
conducted by Pavoni et al., 1972, found out that surface potential reduction was not
solely necessary for microbial flocculation. The polymers might be physically or
electrostatically bond and in the end bridging of the cells of the dispersion into three-
dimensional matrix occurred. The same study supported the polymer bridging theory
in a way that the microbial flocculation increased by the increase in exocellular
polymer to microbial mass ratio.
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12
2.2.2.8. Divalent Cation Bridging Theory (DCB)
Y. Tezuka and R. E. Mc Kinney firstly developed this theory. As can be seen in
Figure 2.4, divalent cations attach anionic parts of the biopolymers and thus form
bridges between the bacterial colonies and flocs, making stable floc matrixes
(Tezuka, 1969; McKinney & Horwood, 1952).
Figure 2.4. Depiction of the DCB Theory
Higgins and Novak, (1997 a ) supported the idea behind this theory, after adding one
of monovalent cations, sodium, floc properties deteriorated due to ion exchange with
divalent cations.
2.2.2.9. Alginate Theory
This theory was first developed by Bruus et al., in 1992. It is known from some of
the studies that Azotobacter sp. and Pseudomonas aeruginosa bacteria is capable of
synthesizing one of the polysaccharides, alginate, in activated sludge systems
(Nunez, et al., 2000; Davies & Geesey, 1995). When the calcium ions are found in
the system, alginate form alginate gels. In this theory it is assumed that the
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13
exocellular polysaccharides are composed of alginates and by the addition of
calcium, binding of particles, calcium and negatively charged alginate surfaces takes
place. Due to this specific type of cation and polymer binding, addition of other
cations just as magnesium and sodium resulted in deterioration in sludge
characteristics by the exchange of ions with calcium (Bruus et al., 1992). Another
study conducted by Sanin and Vesilind in 1996, supported this theory using the
synthetic sludge containing alginate. Magnesium did not give the same result as in
the case of calcium. The working principle of the alginate theory is similar to
divalent cation bridging theory. However DCB is available for any kind of divalent
cation rather than specifically calcium and its binding to alginate specifically.
In 2002 D Sobeck and M. Higgins studied the best fitting model between three
mechanisms by measuring sludge characteristics by adding calcium, magnesium and
sodium concentrations to continuous reactors separately. DCB was found to be the
best fitting model. Floc characteristics of calcium and magnesium showed similar
values, enhancing floc formation. DLVO theory failed due to the deterioration of floc
by addition of the monovalent cation. There was not superiority of calcium reactors
over to magnesium reactors and hence the validity of the alginate theory was lost
partially. The specificity of calcium ions could not be explained by these results.
In addition to all of these theories, it is believed that hydrophilic and hydrophobic
properties of the EPS play a role in bioflocculation mechanism. Higgins and Novak
(1997 a) proposed that hydrophobic parts just as the amino acids of polymers were
essential constituents in activated sludge and biopolymers can bind through
hydrophobic parts. In addition when the hydrophibicity of the floc increased the
sludge became easily settleable since it was easily separated from the hydrophilic,
polar parts (Urbain et al., 1993).
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2.3 Extracellular Polymeric Substances (EPS)
2.3.1 Definition of EPS
Majority of bacteria have polymeric structures, lying outside of their cell walls.
These polymeric structures are comprised of –homo and –heteropolysaccharides,
humic compounds, some types of polypeptides, polyalcohol, lipids and nucleic acids.
Extracellular polymeric substances (EPS) are the common name given to these
polymeric structures (Pavoni et al., 1972; Urbain et al., 1993). They have a
significant role in bioflocculation mechanism.
In addition, EPS has a highly hydrous, gel-like structure and it has an often charged
network into which microorganisms are embedded. In this EPS network, some
dissolved substances in water and particulate substances can be seen due to sticky
nature of the polymer (Sanin et al., 2006).
It can be regarded as these polymers may be in the form of discrete capsule that is
strongly attached to cell wall / surrounding the cell wall or in the form of a jelly-
slime structure that is not strictly adhered to cell wall or not adhered to it They are
released out of the cell wall by active transport or cell lysis (Sutherland, 1972; 1977
and 1990). They have high molecular weight and mass of total EPS constitutes
approximately 80% of the mass of activated sludge (Frolund et al., 1996).
G H. Yu and his co workers in 2008 proposed EPS had different partitions according
to separation principles. When EPS is distributed in bulk solution after settling of
sludge, this portion is named as supernatant EPS. The remaining part that is
embedded in sludge matrix is composed of EPS in slime form and bound EPS
fractions. Slime EPS has loose binding to flocs and can not persist to washout.
Bound EPS, on the other hand, is a discrete covering layer with a distinct margin at
the outside of the cell wall. In addition it is a double layer structure which is named
as loosely bond EPS (LB-EPS) and tightly bound EPS (TB-EPS) based on extraction
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15
procedure (Poxon & Darby, 1997; Ramesh et al., 2006; Li & Yang, 2007; Yu et al.,
2007).
2.3.2 Composition of EPS
As stated before, the extracellular polymeric substances are composed of
polysaccharides, proteins, lipids, nucleic acids and humic substances (Eriksson &
Alm, 1991; Urbain et al., 1993; Frolund et al., 1996). Although these basic
constituents remain same, the dominant composition can change according to
different conditions. There are a number of studies investigating the dominant part of
EPS but there is not an agreement between these results due to the conditions. For
instance, some of the research regarded polysaccharides as the most important
component in EPS taking part in flocculation (Horan & Eccles, 1986; Bruus et al.,
1992, Jorand et al., 1995). On the other hand, others reported that exocellular protein
concentration was higher than the polysaccharide concentration and played the most
important role in flocculation (Tenney & Verhoff, 1973; Brown & Lester, 1980;
Barber & Veenstra, 1986; Urbain et al., 1993).
By the characterization of the EPS, it was found that some portion of the
extracellular polysaccharides was made up of uronic acids (Brown & Lester, 1980;
Forster, 1971; Frolund et al., 1996; Horan & Eccles, 1986). Uronic acids are known
to be polyanionic due to carboxyl group on its surface which determines the basis for
having an interaction with cations present in the system. This property makes the
cations possible to form complexes with extracellular polymeric polysaccharides
(Christensen, 1989; Sutherland, 1990). In addition, same examination was applied
for the exocellular proteins. Proteins were composed of amino acids such as glutamic
and aspartic acid which contain carboxyl group that contributes to negative charge of
bioflocs (Dignac, et al., 1998; Higgins and Novak, 1997 a, b, c).
At neutral pH range, extracellular polymers possess net negative charge that will
soon interact with divalent cations to form bridges (Nguyena, et al., 2008). In
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16
addition strong association between calcium ions and exocellular protein had been
found in some of the studies (Urbain et al., 1993; Higgins and Novak 1997 a).
However due to presence of mixed culture in activated sludge systems it seemed
plausible that there would be differences in uniformity of extracellular metabolites
produced by the culture. In addition, different type of substrate utilization may
change the dominant composition but major components in the EPS structure
remained same (Pavoni et al., 1972).
2.3.3 Factors Affecting EPS Production and Bioflocculation
Many factors affect the EPS composition, EPS production and bioflocculation.
Environmental conditions, genotype and physiological factors among those. The
nutrient composition, C/N, C/P ratio, pH, temperature and agitation speed can be
identified as the environmental factors. These are also essential for the process
efficiency (Salehizadeh & Shojaosadati, 2001).
The physiological state of the microorganisms is one of the factors that affect the
microbial aggregation and EPS production. In his study Pavoni et al., (1972)
discovered that exocellular polymer production and therefore biological flocculation
reached its optimum value when the endogenous growth phase was obtained. The
validity of this data did not change with various biological systems such as aerobic-
anaerobic systems mainly comprising heterotrophic-autotrophic microorganisms
respectively. However in the example of Z. Ramigera, production of the flocculant
terminated after 90h in stationary phase (Norberg & Enfors, 1982). Surprisingly,
flocculation related with S.griseus was not dependent on growth phase
(Shimofuruyauji et al., 1996).
The proportions of the organic concentrations provided in the feed seemed to change
the EPS production and composition. When nitrogen is limited and the carbon
sources are in excess amounts, it has been shown that the cells tend to accumulate
large amounts of various polymers. With a remarkable increase in C/N ratio,
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17
majority of bacteria increase the production of carbonaceous compounds and reduce
the cellular protein and nucleic acid synthesis. Under these conditions, substantial
amounts of EPS are produced (Sanin et al., 2006)
At lower C/N ratio, all carbon sources were utilized by microorganisms in biomass
synthesis and nitrogen was used for protein synthesis. Microorganisms probably did
not tend to produce extracellular polysaccharides. However higher amounts of
extracted proteins were observed which can function inside the cell or found in the
extracellular medium (Sanin et al., 2006).
Another operational parameter, mean cell residence time (MCRT), had an influence
on the EPS production. According to previous studies, larger amounts of EPS were
produced due to endogenous metabolism of microorganisms at higher MCRTs than
lower MCRTs (Pavoni et al., 1972; Chao & Keinath, 1979; Steintuch, 1987).
At higher MCRT values the protein component predominated. At low MCRT (high
F/M), there is high quantity of food. However microorganisms do not have the
sufficient time to consume most of the carbon source. They tend to convert excess
carbon source to EPS. At higher MCRT, food is in lower quantities; microorganisms
utilize it in biomass synthesis. They just secrete the EPS to outside which could be
used for microbial protection. At the stationary growth phase of microorganisms, cell
lyses takes place and therefore abundance of proteins at higher MCRTs might be
explained by this reason (Sanin et al., 2006). As MCRT increases, total EPS amount
increases. By an increase in MCRT; protein component of extracted EPS increases.
On the other hand, there is not a significant change in carbohydrate content (Sanin et
al., 2006).
Since the system is dependent upon the microorganisms behavior temperature ranges
are also significant. There is an optimum temperature range for obtaining higher
concentration of EPS. Mixed culture R-3 (Kurane & Matsuyama, 1994), Bacillus sp.
PY-90 (Yokoi et al., 1995), Flavobacterium sp. (Endo et al., 1976), Bacillus sp. DP-
152 (Suh et al., 1997) were reported to produce optimum EPS concentration at a
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18
temperature of 30°C whereas Zoogloea MP6 was able to produce EPS at 20°C (Kakii
et al., 1996).
As well as the temperature, pH affected EPS production. The optimum pH range for
the EPS production varied for different species. For instance, C.xerosis could
produce EPS at lower pH (Esser & Kues, 1983) whereas A. Sojae produced
biopolymers in the alkaline pH.
2.4 Cations
Main cations that are found in activated sludge systems are sodium, potassium,
ammonium, calcium, magnesium, iron and aluminum. Ions have three fundamental
functions in the cell:
i) Being coenzymes or metal cofactors
ii) Transferring of electrons in oxidation-reduction reactions
iii) Serving as regulators of osmotic pressure (Gerardi et al., 1994).
Besides this, they are regarded as one of the major components of the activated
sludge flocs as well as the microorganisms and extracellular polymeric substances
(Bruus et al., 1992; Higgins and Novak, 1997 a).
It was known from previous studies that exocellular biopolymers and cations took
part in flocculation process. According to some of the bioflocculation mechanisms,
negative sites on exocellular biopolymers were binded by the help of cations and
therefore supported flocculation (Tezuka, 1969; Novak & Haugan, 1978; Bruus et
al., 1992). Not having a proportional cation concentration in the feed would lead to
formation of weak and dispersed flocs (Park, 2002).
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19
2.4.1 Divalent Cations
Many researches have been conducted for understanding the role of divalent cations
on flocculation. Calcium and magnesium might be needed for adhesion of certain
monocultures of certain bacteria (Tezuka, 1969; Lodeiro et al, 1995).
A floc model in which divalent cations were involved has been proposed (Higgins
and Novak, 1997 b). Lectin like proteins that were attached to bacterial surface cross-
linked with polysaccharides by the help of divalent cations (calcium and
magnesium). Network was formed by binding the exocellular polysaccharides and
proteins. The illustration of the floc model can be seen in Figure 2.5
Figure 2.5 Roles of Biopolymers and Divalent Cations on
Bioflocculation(B:Bacteria,LLP:Lectin-like protein,P:polysaccharide,C++:Divalent
cation (Higgins and Novak,1997 b)
The removal or extraction of calcium ions by using EGTA (Bruus et al., 1992),
EDTA (Kakii et al., 1985) and cation exchange resin (CER) (Keiding & Nielsen,
1997) led to increase of turbidity and worsening of filterability and settleability. At
the same time, the study conducted by Bruus et al.. in 1992 ,indicated that the
extracellular polymers may be alginate or another polysaccharide whose properties
resemble those of alginate and form a gel-like structure in the presence of calcium
ions. Further, another study conducted by Sanin and Vesilind in 1996, enhanced this
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20
proposal by forming stable synthetic sludge flocs due to the addition of calcium and
alginate into a suspension of stable particles.
Magnesium’s role on bioflocculation was studied as well. Higgins and Novak, (1997
a) observed in their study that the system required calcium and magnesium for
acceptable physical characteristics whereas the same system required the addition of
magnesium in the second trial. Further study revealed the same system was
dominated by different type of bacteria at different time intervals and therefore the
requirements for the cations altered. It was concluded in some of the studies the
change of the physical characteristics of the system to the absence of calcium and
magnesium depended on the type of the bacteria dominant in the system (Tezuka,
1969; Endo et al., 1976; Lodeiro et al., 1995). Higgins and Sobeck (2002) found that
sludge settling and dewaterability characteristics were enhanced by addition of either
magnesium or calcium.
The effect of the divalent cations differed on the sludge characteristics although they
have similar properties in some of the studies. For instance, by the addition of
calcium ions, the bound water content was reduced in an earlier study. On the other
hand magnesium ion had no such an effect on the bound water content (Forster and
Lewin, 1972). The same study had also found out that the extracellular polymers
would rather prefer calcium ions than magnesium ions to have interactions.
Binding ability of the components of the extracellular biopolymers to divalent
cations is another important issue that has been discussed. It was found that by the
addition of calcium and magnesium ions, bound protein concentration of the EPS
increased (Urbain et. al., 1993; Dignac et al., 1998, Higgins and Novak, 1997 a).
On the contrary one of the studies conducted by Nguyena et al., (2008) supported the
previous findings that divalent cation concentration caused a decrease in supernatant
EPS carbohydrate concentration in semi-continuous and batch reactors. In same
manner, the bound EPS carbohydrate concentration increased by increasing calcium
concentration.
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2.4.2 Monovalent Cations
In activated sludge systems, higher concentratios of sodium resulted in the
deterioration in settling and dewaterability of sludge (Novak and Randall, 1986;
Bruus et al., 1992). Up to 5meq/l of sodium SVI decreased .Above 5 meq/L sludge
volume index (SVI) which is an indicator of sludge settleability increased and up to
20 meq/L, the system had reached such a state that SVI could not be measured due to
deflocculation (Higgins and Novak, 1997 a).
Due to displacement of divalent cations within the floc with high sodium
concentrations according to ion exchange process, poor settling and dewatering took
place. Floc structure weakened due to removal of divalent cations, cation bridging
function destroyed (Tezuka, 1969; Novak and Haugan, 1978; Bruus et al., 1992).
When the soluble cation concentrations of magnesium, calcium and sodium were
measured after the addition of sodium, it was observed that sodium brought about an
increase in soluble calcium and magnesium concentration meaning that release of
calcium and magnesium from biopolymer network due to sodium (Higgins and
Novak, 1997 a).
When another monovalent cation, potassium concentration amount was increased,
SVI or settling properties improved due to formation of very large flocs which was
capable of settling rapidly. On the other hand dewatering properties got worse
(Higgins and Novak, 1997 a).
As it was explained before, cation bridging mechanism is one of the mechanisms of
bioflocculation. Ionic charge, ion size and radius of hydration shell of the cations are
the factors that influence on the binding ability of cations. It is known that cations
with higher valency, large size and thin hydration shell may easily get closer to
charged sites of surfaces and form bonds with negative charged sites of EPS (Piirtola
et al., 1999). Among four cations, magnesium, calcium, sodium and potassium,
potassium has the lowest hydration shell radius (Mg2+ > Ca2+>Na+>K+). Due to this
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22
fact, potassium becomes more easily dehydrated, moves to charged sites of EPS and
forms stronger bonds with these sites compared to sodium ions. One of the studies
conducted by Rengasamy and Naudi (1998), found out relative flocculation power of
cations within the following order: Ca2+> Mg2+ > K+ > Na+. It can be derived from
this data that sodium is the weakest flocculator due to larger hydrated shell radius,
single charge and small size.
In addition to this, the ability of the cations to be incorporated in floc matrix
depended on the functions of the cations in the cell. It was known that potassium ions
were responsible for maintaining osmotic pressure in the cell (Gerardi et al., 1994).
Therefore it acted in the intracellular medium rather than magnesium and calcium
ions which were in extracellular medium and helped in binding of flocs (Sanin et al.,
2006). As it happened in the sodium case, another monovalent cation ammonium
caused deterioration in floc characteristics (Novak, 2001).
2.4.3 Monovalent ion / divalent ion (M/D) Ratio
Higgins and Novak, (1997 a) found out that, physical properties of the sludge got
worse when the proportion of monovalent to divalent cations were greater than 2.
Therefore they regarded M/D as an indicator to determine for floc characteristics as
well as the amount of individual concentrations of the cations.
2.4.4 Trivalent Cations
There were not enough investigations that focused on the concentration of trivalent
cations on activated sludge systems although these cations were having higher
concentrations in these systems. At an acid treatment of the activated sludge systems
near a pH value of 3, iron and aluminum could not be extracted from the sludge
matrix whereas calcium and magnesium could be extracted (Kakii et al., 1985). In
other studies it was shown that ferric iron had high affinity for binding protein in
sludge (Murthy et al., 2000; Muller, 2001).
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23
The number of the studies investigating the effect of aluminum which was
commonly used as coagulants for removing natural organic matters was limited. One
of the studies found out that alum was able to remove humic substances from the
swamp water at pH of 5 to 7. Since the humic substances are one of the major
constituents of EPS there was the possibility of having an interaction between EPS
and alum (Lu et al., 1999).
Nguyena et al (2008) researched the contribution of a trivalent cation with calcium
ion to flocculation. The samples containing both calcium and alum had higher
flocculation than the samples containing only calcium. Use of these cations increased
the bound polysaccharide content as expected.
In the study conducted by Yu et al (2009) it was observed that in tightly bound EPS
extracted samples, the contribution of trivalent cations to sludge matrix was higher
than calcium and magnesium ions.In another study the availability of iron and
aluminum caused improvement of activated sludge settling in a way by reducing
number of the filamentous bacteria and preventing bulking (Agridiotis et al., 2007).
2.5 Activated Sludge Properties Related To Bioflocculation
2.5.1 Dewaterability
The conversion of organic compound to less harmful inorganic compounds in
activated sludge process is not enough for the process efficiency. The biosolids
should be separated from the system by settling. In addition the settled sludge or the
sludge that is going to be wasted should release its water content easily and hence
should not contain higher amounts of water in its content. Since the thickened sludge
is only 3-5 % solids when there are difficulties in dewaterability, the cost of sludge
handling increases dramatically. Therefore it is true to conclude that sludge
dewatering is the bottleneck of the sludge handling operation (Karr and Keinath,
1978).
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24
The dewaterability of sludge is generally determined by identifying rate of filtration
and bound water content of the sludge. In sludge, water is composed of “bound”
water and “free” water (Lee et al., 1994, 1995; Wu et al., 1998). The latter one is
easy to remove from sludge by mechanical means whereas the former one is held in
floc matrix, bound to sludge particles firmly and hence cannot be removed by simple
mechanical means (Lee et al., 1994; Colin et al., 1995). Bound water can be defined
as sum of water hold in capillaries and voids inside the sludge flocs and between
them (interstitial water), internal water in bacterial cells and water
chemically/physically bound in the sludge and surface water that is adsorbed to wet
sludge surface (Jin et al.,2004).
The dewaterability of sludge can be assessed by applying several analyses. Capillary
suction time (CST) measurements, specific resistance to filtration (SRF) and bound
water measurements are some of those. Specific resistance to filtration was first
developed by Coakley et al. (1956). By applying Darcy’s equation, through a porous
medium pressure drop for a flow was analyzed. Although there have been some
modifications of the technique and image of the SRF over the years (Christensen and
Dick, 1985) the measurements were not successful in estimating the actual vacuum
or pressure filter performance (Vesilind, 1988). Moreover it was time-consuming
(Novak and Knocke, 1987).
Due to its practical use, CST measurements are generally preferred in activated
sludge systems. It measures time that is required for the sludge to permeate through
the filter paper for a constant distance. For the studied specific sludge sample, CST
and rheological properties of sludge depended on suspended solids concentration
(Mikkelsen and Keiding, 2002). Higher values of the CST are the indication of poor
dewaterability and filterability (Smollen, 1990; Lin et al., 1996; Higgins and Novak,
1997 a, b; Murthy and Novak, 1999; Lee and Liu, 2000).
Floc characteristics and floc properties affect the dewaterability of the sludge
(Karrand et al., 1978; Novak et al., 1988; Sorensen et al., 1995). Flocs contain
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extracellular polymeric substances, organic and inorganic molecules as well as
microorganisms (Frolund et al., 1996). One of these constituents, EPS, is able to bind
a portion of the water in the sludge although major part of the water is trapped by cell
walls in order to be used intracellular (Keiding et al., 2001; Eriksson et al., 1992).
Since EPS and its network are negatively charged, the osmotic pressure would cause
high amount of water uptake into the floc matrix (Keiding et al., 2001). As a result
there is a strong relation between EPS concentration and dewaterability (Houghton et
al., 2001; Mikkelsen et al., 2002).
According to the study conducted by Mikkelsen and Keiding (1995), by the increase
in concentration of total extractable EPS, CST of the sludge decreased. This result
might be obtained due to the fact that easily extractable EPS binds flocs and water in
the bulk sludge weakly. As a result smaller values in CST, which is found to be
mostly related with free water content in the bulk sludge (Jin et al., 2004), are
obtained in analysis. On the contrary, Kang et al., (1989) indicated that by the
addition of extracted EPS to system, dewatering characteristics decreased. In the
study carried out by Sanin et. al. (2000), the extraction of calcium ions caused the
release of extracellular polymers and therefore induced deflocculation. In addition,
the filterability decreased. Some of the studies concluded that a certain concentration
of EPS was required for enhancing filterability of sludge (Durmaz and Sanin, 2003;
Houghton et al., 2001). However, after exceeding this certain amount of
concentration, the dewaterability decreased due to the ability of EPS matrix for
trapping of the water (Houghton et al., 2001).
The EPS composition influences the dewaterability as well as the EPS concentration.
Water binding depended on the hydrophilic interactions and hydrogen bonding. By
the contribution of other molecules such as the –OH and –NH bonds with hydrogen
bond, water bonds were formed. In this sense, proteins and polysaccharides of the
EPS contribute to water binding (J. Schmitt et al., 1999; Jin et al., 2004). There is
some contradiction about positive or negative correlation of these constituents.
Bowen and Keinath (1984) found that carbohydrate, protein and surface lipids had
positive impact on dewaterability. Protein component of the EPS was found to be
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more hydrophobic and hence became easily separated from the hydrated media and
filterability eased (Sanin and Sesay, 2004). This result was in agreement with some
of the studies (Sanin et al., 2006, Jin et al., 2004; Higgins and Novak, 1997 a, b, c).
On the contrary, elevation of CST and difficulty in dewaterability of the sludge was
observed by the release of protein to solution (Murthy and Novak, 1998, 1999;
Novak et al., 2001).
Higher concentrations of more hydrophilic component, EPS carbohydrate, resulted in
deteriorations of the dewaterability (Sanin et al., 2006; Murthy and Novak, 1999).
In addition to EPS characteristics and concentration, the particle size distribution has
a significant impact on dewaterability. In the case of chemical coagulants, the
effectiveness of the coagulation depended on the capability of the coagulants to
increase the particle size (EPA, 1974). It was found out from the investigation of
particle size distributions in activated sludge treatment systems, most influential
particle size belonged to supracolloidal particles in the range of 1 to 100µm. There
was negative correlation between the increase of the concentration of the particles in
this size range and dewaterability. This size range caused the clogging of the filter
medium and sludge cake (Karr and Keinath, 1978). On the other hand, Novak et al.,
(1988), found out that particle size smaller than 40 µm resulted in poor dewatering.
There are number of studies regarding the effect of cations on dewaterability. Due to
presence of divalent cation bridging mechanism the flocculation eases with the
addition of cations such as calcium and magnesium. As a result the dewaterability of
the sludge improved (Higgins and Novak, 1997 a). The presence of trivalent cations
such as aluminum and iron enhanced dewaterability as well (Keiding and Nielsen,
1997; Knocke et al., 1996; Colin and Gazbal, 1995; Higgins and Novak, 1997).
Apart from the multivalent cations, monovalent cations seemed to affect sludge
dewaterability negatively. Especially higher concentrations of the sodium resulted in
bad filterability (Novak and Randall, 1986; Bruus et al., 1992). This result was
attributed to the displacement of monovalent cations with divalent cations that the
bridging between floc components destroyed. The presence of the monovalent
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cation, potassium resulted in poor dewaterability as traced by SRF values whereas
the settleability was not affected (Higgins and Novak, 1997 a).
2.5.2 Settleability
The effective settling of the activated sludge biosolids is one of the key factors for
effective treatment in activated sludge systems. In the case of ineffective settling, the
quality of the effluent gets worse. Therefore settling characteristics is crucial for the
success of the overall system.
Settling of the system is determined by Sludge Volume Index (SVI) and Zone
Settling Velocity (ZSV). SVI is defined as the volume in milliliters occupied by 1g
of suspension after 30 minutes of settling (Dick and Vesilind, 1969). SVI value
bigger than 150 is taken as indication of deterioration in settling (Jenkins et al.,
1993).
Many factors have been identified to affect the settling. These are listed as organic
loading of the system (Sürücü, 1982; Chao and Keinath, 1979; Barahona and
Eckenfelder, 1984), mean cell residence time (Bisogni and Lawrence, 1971; Sürücü,
1982; Chao and Keinath, 1979), physical, chemical nature of the floc surface and
physiological and biochemical nature of the flocs (Forster et al.,1976; Smith and
Novak, 1982; Lee, and Ganczarczyk, 1986;Horan and Shanmugan, 1986). As a result
there is a strong relationship between the settleability and flocculation.
Physiological and biochemical nature of flocs and therefore settleability seemed to be
strongly affected by pH, temperature and dissolved oxygen concentration (Sanin and
Sürücü, 1989). When the sludge temperature increased from 15 to 35°C, settleability
decreased due to structural changes in polysaccharidic and proteinaceous part of the
EPS. In addition, increasing the pH of the system lead to an increase in settleability
since the addition of the anions caused an increase in reactive sites of the EPS and
enhanced flocculation. Low concentrations of DO caused highly turbid supernatant
and not properly measured settleability parameters (Sanin and Sürücü, 1989).
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There are different findings of different studies concerning the EPS concentration
and settleability. When the EPS concentration increased, zeta potential increased due
to negativity of EPS. Then the electrostatic repulsion forces between negatively
charged floc components became stronger as in the case of DLVO theory. This
resulted in poorer compaction and settling (Zita and Hermansson, 1994). Increase of
the EPS concentration had negatively affected flocculation (Wilen et al., 2008).
Higher concentration of the biopolymers avoided the flocculation of bacterial mass
and caused poor settling (Harris and Mitchell, 1975; Kakii et al., 1989; Urbain et al.,
1993; Liao et al., 2001).
The dominant component of the EPS also affected the settleability in some of the
studies. Higgins and Novak, (1997 a), had positively correlated the bound protein
concentration and settleability. Liao et al. (2001) and Urbain et al. (1993) found
similar results indicating the improvement of settleability with higher protein
content.
Increased polysaccharide concentration seemed to negatively influence settleability
of the sludge (Urbain et al. 1993, Randall et al., 1971; Forster, 1971). In the same
manner a high C/N ratio of 43 caused an increase of the carbohydrate portion of EPS
under nutrient deficient conditions. This led to an increase in SVI, indicating poor
settleability and sludge bulking condition (Durmaz and Sanin, 2001).
Divalent and monovalent cations played role in settleability. Bruus et al. (1992) and
Higgins and Novak (1997 b) indicated that excess concentrations of the monovalent
cations caused deterioration in floc structure and settleability. However this
conclusion was not same for all the monovalent cations. Excess sodium caused poor
dewaterability, settling and bioflocculation (Higgins and Novak, 1997 a) whereas
potassium led to worsening of dewaterability while there was not a problem in
settling (Novak et al., 1996; Higgins and Novak, 1997 a).
Addition of the divalent cations such as calcium and magnesium improved sludge
settling proving the validity of the divalent cation bridging mechanism. Moreover
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monovalent/divalent ratio that is bigger than 2 caused problems in settling (Higgins
and Novak, 1997 a).
Moreover floc density and floc particle size affected the settleability. Stoke’s law
explained the relationship between the floc size and density and settleability. By the
increase of the floc density and floc size the settling properties improved. Plants
reported good settling properties have floc densities in the range of 1.025 – 1.035
g/mL range. The floc density and floc size was shown to be one of the main factors
influencing the settling characteristics (Higgins and Novak, 1997 a and Murthy and
Novak, 1997).
2.5.3 Rheology
Rheology can be described as the science concerning about the relationship between
stress and deformation (strain). One of the rheological parameters, viscosity, is used
for understanding the “fluidity” of a fluid (Young et al., 1994). It can be mainly
described as the “resistance of the fluid to flow” or the proportionality constant
between the shear stress and shear rate of a fluid element which can be formulated as
below:
(2.1)
Where τ is the shear stress, du/dy is the shear rate. When there is a linear relationship
between the shear stress and the shear rate the fluid is said to be Newtonian fluid
which can be formulated as below:
(2.2)
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Here, the definition for the viscosity, µ, is valid that viscosity is proportionality
constant. Newtonian viscosity is constant under a certain temperature and pressure
(Hou and Li, 2003). However activated sludge is known to obey non-Newtonian
behavior (Hou and Li, 2003; Sozanski et al., 1997). The viscosity of sludge depends
on a shear rate gradient under certain pressure and temperature (Dentel, 1997;
Spinosa and Lotito, 1997).
The ratio of shear stress to shear strain gives the apparent viscosity in non-Newtonian
fluids (Young et al., 1994). Apparent viscosity in sludge is the reflection of the
internal and external forces acting within the sludge flocs and also it indicates the
deformation of the floc under stress (Dentel et al., 1997; 2000).
Figure 2.6 The Rheograms of Different Fluids (Vesilind, 1979)
There are different types of Non-Newtonian fluid behavior as can be seen in Figure
2.6 above. 1 denotes Bingham plastic; 2 represents Pseudo-plastic; 3 is the
Newtonian fluid and 4 is the dilatant fluid.
The Bingham plastic shows neither fluidity nor solidity. Up to a certain point with a
finite shear stress the material does not move. However once the yield stress is
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exceeded the fluid starts to flow (Young et al., 1994). Bingham plastic behavior is
formulated as follows:
(2.3)
Where τy= yield stress and ƞ is the plastic viscosity.
According to the studies, activated sludge rheology was mostly reported to be the
pseudo plastic (Lotito and Spinosa, 1997; Moeller and Torres, 1997; Proff and
Louhmann, 1997; Krauth and Staab, 1992).This behavior is formulated as follows:
(2.4)
Where K= fluid consistency index and n= flow behavior index w